Cancer is a leading cause of morbidity and mortality of the human population. Considerable evidence suggests that environmental agents may be the principal causes of human cancers. The long-term objective of this project is to investigate the role of mitochondria-derived reactive oxygen species (ROS) in environmental agent-induced tumor promotion. The hypothesis we are currently investigating is that induction of ROS from mitochondria through protein kinase C (PKC)-mediated signal transduction is a mechanism of chemically induced tumor promotion. Accordingly, the specific aims of this proposal are designed to (1) investigate the mechanisms underlying PKC-induced production of mitochondria-derived ROS in cultured epidermal cells, and (2) determine the involvement of mitochondria-derived ROS induced by PKC signaling in molecular and biochemical alterations leading to tumor promotion in vitro. To achieve these goals, mouse keratinocyte cell line MEL-30 and the epidermal JB6 cells, a popular in vitro tumor promotion model, will be used. The widely studied PKC-activating tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate (TPA) will be utilized as a model chemical to investigate the above hypothesis. Biochemical and molecular techniques will be applied to determine the PKC isozyme(s) responsible for the induction of mitochondrial ROS and to examine the role of PKC-induced mitochondria-derived ROS in anchorage-independent growth of JB6 cells as well as activation of cellular factors that are believed to be critically involved in tumor promotion. Fulfillment of the above aims will result in a greater understanding of the involvement of mitochondria-derived ROS in tumor promoting activities of not only phorbol esters, but other PKC-activating agents, such as asbestos, cigarette smoke, estrogens, polychlorinated biphenyls, and ultraviolet light. Because tumor promotion is the rate-limiting step in multistage carcinogenesis, knowledge of the mechanisms underlying tumor promotion will contribute to our ability to predict risk and to develop possible chemoprotective and treatment regimes against environmental agent-induced cancers.